The subject invention is directed generally to the human platelet F11 receptor, and more particularly to nucleic acid molecules encoding the human platelet F11 receptor and uses thereof.
Throughout this application various publications are referenced, many in parenthesis. Full citations for each of these publications are provided at the end of the Detailed Description. The disclosures of each of these publications in their entireties are hereby incorporated by reference in this application.
The significant role of platelet activation in hemostasis is well-documented. Platelet activation is necessary for platelet aggregation and secretion, and is initiated by the binding of agonist to receptors at the platelet surface. Over the last ten years, several laboratories have developed monoclonal antibodies to platelet membrane glycoproteins. These platelet membrane glycoproteins can serve as agonist receptors on the platelet membrane. The antibodies to these glycoproteins have been of great value in studies designed to elucidate the structure and function of these glycoproteins.
Most of the success in raising monoclonal antibodies to platelet receptors was in studies on the integrin cohesion receptor IIb/IIIa (Coller et al., 1983; Kornecki et al., 1984) and the adhesion receptor Ib/IX (Coller et al., 1983; Handa et al., 1986). Functional antibodies that inhibit the action of these receptors provided a large body of new information and have led to direct conclusions about the functions of these glycoprotein receptors. Such inhibitory antibodies were also shown to have potential in vivo therapeutic use (Coller et al., 1986; Peters et al., 1986).
Some of these antibodies that serve as agonists, binding to the receptors at the platelet surface and thereby activating the platelets, have been identified. Several laboratories have developed or identified such xe2x80x9cactivatorxe2x80x9d antibodies that appear to react with platelet membrane protein components of 21-24 kD. The first report of a monoclonal antibody which served as an agonist and induced platelet aggregation was published by Boucheix et al. in 1983. This monoclonal antibody immunoprecipitated a platelet protein with apparent molecular weight (M.W.) of 24 kD under both reduced and non-reduced conditions. The addition of Fab fragments of this antibody to platelets resulted in the inhibition of platelet aggregation induced by various agonists. The platelet antigen recognized by this antibody was identical to the leukemia-associated antigen, p24, found in common acute lymphoblastic leukemia cells and neuroblastoma cells (Kersey et al., 1981; Jones et al., 1982; Komada et al., 1983).
Thiagarajan et al. (1983) reported that platelet aggregation could be induced by another monoclonal antibody. This antibody was found to be directed against a 21 kD protein present in both normal and Glanzmann""s thrombasthenic platelets.
Gorman et al. (1985) have described several monoclonal antibodies which induce platelet aggregation. All of these antibodies immunoprecipitated a 24 kD platelet protein in both the reduced and non-reduced states. The Fab fragments of these antibodies were found to augment the aggregation of platelets by adenosine diphosphate (ADP).
Higashihara et al. (1985) also described a monoclonal antibody which induced platelet aggregation and secretion by interaction with a protein of 24 kD. Preincubation of platelets with this antibody inhibited ristocetin-induced agglutination. It is known that these antibodies are directed against the p24/CD9 protein on the platelet surface. The CD9 antigen has been cloned and sequenced (Boucheix et al., 1991; Lanza et al., 1991), and CD9 antibodies have been shown to induce platelet aggregation mediated by the FcyRII receptor (CD32 molecule) (Worthington et al., 1990).
Duncan and Rosse (1986) showed that antibodies to platelet HLA class I antigen (anti-ABH IgG) could activate platelets and induce serotonin release. Similar results were obtained by Cosgrove et al. (1988), who reported that three different anti-HLA Class I monoclonal antibodies and an anti-xcex22 microglobulin antibody caused platelet aggregation and secretion. Duncan and Rosse (1986) also showed that high concentrations of anti-PLA1 antibodies inhibited platelet secretion induced by these antibodies. Ryu et al. (1989) found that high concentrations of PLAl blocked fibrinogen binding resulting in the blockage of agonist-induced platelet aggregation, whereas low concentrations of anti-PLA1 antibodies induced release and aggregation.
Activator monoclonal antibodies directed against GPIIb and GPIIIa have also been reported. A stimulatory monoclonal antibody to the GPIIb/IIIa complex has been described by Modderman et al. (1988) which induces the release of alpha and dense granule contents resulting in platelet aggregation. Morel et al. (1989) have described a monoclonal antibody directed against GPIIb. The F(abxe2x80x2)2 fragments of this antibody did not induce platelet aggregation although they blocked the stimulation of platelets by the intact antibody.
In addition to these antibodies, antibodies of other specificity have been described which activate platelets. Scott et al. (1989) described a monoclonal antibody which stimulates platelet secretion and aggregation and is directed against a platelet membrane glycoprotein of M. W. 67 kD. Recently, Yanabu et al. (1991) detected an autoantibody in a patient with immunothrombocytopenia (ITP), which activated normal platelets by interacting with a 36 kD platelet surface protein.
Kornecki et al. (1990) referred to a monoclonal antibody called M.Ab.F11 which induces vesicular secretion and aggregation in human platelets. U.S. Pat. No. 5,665,701, issued Sep. 9, 1997, of Kornecki et al. (the entire contents of which are incorporated herein by reference) discloses further details of the F11 receptor protein, including partial amino acid sequences. Although the Patent refers to DNA encoding the platelet membrane glycoprotein F11 (column 4, line 10), no nucleotide sequences encoding the protein are provided or described.
The health related significance of these antibodies which can activate human platelets is apparent. Characterization of the antigens which serve as receptors for these antibodies in the activation process is necessary as well as the elucidation of the biochemical pathways triggered by these interactions.
To this end, the subject invention provides an isolated nucleic acid molecule encoding a human platelet F11 receptor. The invention also provides an antisense nucleic acid molecule complementary to at least a portion of the mRNA encoding the human platelet F11 receptor.
The isolated nucleic acid molecules of the invention can be inserted into suitable expression vectors and/or host cells. Expression of the nucleic acid molecules encoding the human platelet F11 receptor results in production of human platelet F11 receptor in a host cell. Expression of the antisense nucleic acid molecules in a host cell results in decreased expression of the human platelet F11 receptor.
The invention further provides a ribozyme having a recognition sequence complementary to a portion of mRNA encoding a human platelet F11 receptor. The ribozyme can be introduced into a cell to also achieve decreased expression of human platelet F11 receptor in the cell.
The invention further provides a method of screening a substance for the ability of the substance to modify F11 receptor function, and a method of obtaining DNA encoding a human platelet F11 receptor.
Further provided is an isolated nucleic acid molecule encoding a human platelet F11 receptor, wherein the nucleic acid molecule encodes a first amino acid sequence having at least 90% amino acid identity to a second amino acid sequence. The second amino acid sequence has an amino acid sequence selected from the group consisting of SEQ ID NO:3, amino acid residues 28-299 of SEQ ID NO:3, SEQ ID NO:4, and amino acid residues 28-193 of SEQ ID NO:4.
The invention further provides a DNA oligomer capable of hybridizing to a nucleic acid molecule encoding a human platelet F11 receptor. The DNA oligomer can be used in a method of detecting presence of a human platelet F11 receptor in a sample, which method is also provided by the subject invention.
The subject invention is based on the discovery that a stimulatory monoclonal antibody (termed M.Ab.F11) induces aggregation and granule secretion of human platelets through its specific binding to a platelet membrane glycoprotein duplex of MW 32 and 35 kDa, termed F11 receptor (also referred to as FAM or F11-Adhesion Molecule). Internal amino acid sequences of FAM enabled the cloning of a full-length cDNA. The predicted amino acid sequence revealed that FAM is an integral membrane protein. An extracellular N-terminal region consisting of two Ig-like C2-type domains is followed by a transmembrane domain and a short cytoplasmic tail. FAM is thus a new member of the Ig gene superfamily. FAM showed 69% homology with the murine junctional adhesion molecule (JAM). An immunocytochemical location in the tight junctions of HUVEC cells, similar to that shown by JAM in mouse cells, suggests a role for FAM in cell-cell adhesion in addition to participation in platelet activation. By Western blotting, a number of myeloid and vascular cells were,found to express FAM. The detection of autoantibodies against FAM in the circulation of thrombocytopenic and renal patients indicates that FAM may play a pathophysiological role in clinical disorders involving the activation of platelets in vivo.